Microorganisms and methods for overproduction of DAHP by cloned PPS gene

ABSTRACT

Genetic elements comprising expression vectors and a gene coding for phosphoenol pyruvate synthase is utilized to enhance diversion of carbon resources into the common aromatic pathway and pathways branching therefrom. The overexpression of phosphoenol pyruvate synthase increases DAHP production to near theoretical yields.

This is a request for filing a Continuation under 37 C.F.R. §1.60 ofprior Ser. No. 08/801,454 filed on Feb. 18, 1997, now U.S. Pat. No.5,906,925, and of prior Ser. No. 09/277,183 filed on Mar. 26, 1999, tobe issued as U.S. Pat. No. 5,985,617, both of JAMES C. LIAO forMICROORGANISMS AND METHODS FOR OVERPRODUCTION OF DAHP BY CLONED PPSGENE.

GOVERNMENT RIGHTS

This work was supported in part by the National Science Foundation(Grant BCS-9257351), the Welch Foundation (Grant A-1251), and the TexasHigher Education Coordinating Board (Grant 999903-084). The UnitedStates Government may own non-exclusive rights in and to the invention.

FIELD OF THE INVENTION

The present invention relates to the biosynthetic production of organicchemical compounds. In particular, the present invention relates tomethods for increasing the yield of 3-deoxy-D-arabino-heptulosonate7-phosphate (DAHP) in microorganisms through genetic alterations. Thepresent invention also relates to methods of enhancing the production ofcyclic and aromatic metabolites derived from DAHP in microorganismsthrough genetic alterations. For example, the biosynthesis of DAHP isthe first step in the common aromatic pathway from which tyrosine,tryptophan, phenylalanine, and other aromatic metabolites are formed.Also, pathways branching from the common aromatic pathway provide suchuseful chemical products as catechol and quinoid organics such as quinicacid, benzoquinone, and hydroquinone. In addition, aspartame and indigocan be produced from products derived from the common aromatic pathway.

BACKGROUND OF THE INVENTION

Production of chemicals from microorganisms has long been an importantapplication of biotechnology. Typically, the steps involved indeveloping a microorganism production strain include (i) selection of aproper host microorganism, (ii) elimination of metabolic pathwaysleading to by-products, (iii) deregulation of such pathways at both theenzyme activity level and the transcriptional level, and (iv)overexpression of appropriate enzymes in the desired pathways.

The last three steps can now be achieved by use of a variety of in vivoand in vitro methods. These methods are particularly user-friendly inwell-studied microorganisms such as Escherichia coli (E. coli).Therefore, many examples of engineered microorganisms for physiologicalcharacterization and metabolite production have been published.

In most cases, the first target for engineering is the terminal pathwayleading to the desired product, and the results are usually successful.However, further improvements of productivity (product formation rate)and yield (percent conversion) of desired products call for thealteration of central metabolic pathways which supply necessaryprecursors and energy for the desired biosyntheses of those products.

Cyclic and aromatic metabolites such as tryptophan, phenylalanine,tyrosine, quinones, and the like trace their biosynthesis to thecondensation reaction of phosphoenolpyruvate (PEP) and D-erythrose4-phosphate (E4P) to form DAHP. DAHP biosynthesis is the first committedstep in the common aromatic pathway. DAHP biosynthesis is mediated bythree DAHP synthases or isoenzymes. These isoenzymes are coded by genesaroF, aroG, and aroH, whose gene products are feed-back inhibited bytyrosine, phenylalanine and tryptophan, respectively.

After DAHP biosynthesis, some DAHP is converted to chorismate.Chorismate is an intermediate in biosynthetic pathways that ultimatelyleads to the production of aromatic compounds such as phenylalanine,tryptophan, tyrosine, folate, melanin, ubiquinone, menaquinone,prephenic acid (used in the production of the antibiotic bacilysin) andenterochelin. Because of the large number of biosynthetic pathways thatdepend from chorismate, the biosynthetic pathway utilized by organismsto produce chorismate is often known as the “common aromatic pathway”.

Besides its use in chorismate production, DAHP can also be converted toquinic acid, hydroquinone, benzohydroquinone, or catechol as describedby Draths et al. (Draths, K. M., Ward, T. L., Frost, J. W.,“Biocatalysis and Nineteenth Century Organic Chemistry: conversion ofD-Glucose into Quinoid Organics,” J. Am. Chem. Soc., 1992, 114,1925-26). These biosynthetic pathways branch off from the commonaromatic pathway before shikimate is formed.

The efficient production of DAHP by a microorganism is important for theproduction of aromatic metabolites because DAHP is the precursor inmajor pathways that produce the aromatic metabolites. The three aromaticamino acids, besides being essential building blocks for proteins, areuseful precursor chemicals for other compounds such as aspartame, whichrequires phenylalanine. Additionally, the tryptophan pathway can begenetically modified to produce indigo.

The production of tryptophan and phenylalanine by E. coli has been welldocumented. For example, Aiba et al. (Aiba, S., H. Tsunekawa, and T.Imanaka, “New Approach to Tryptophan Production by Escherichia coli:Genetic Manipulation of Composite Plasmids In Vitro,” Appl. Env.Microbiol. 1982, 43:289-297) have reported a tryptophan overproducerthat contains overexpressed genes in the tryptophan operon in a hoststrain that is trpR and tna (encoding tryptophanase) negative. Moreover,various enzymes, such as the trpE gene product, have been mutated toresist feedback inhibition. Similar work has been reported forphenylalanine production.

In the past, the enhanced commitment of cellular carbon sources enteringand flowing through the common aromatic pathway has been accomplishedwith only modest success (i.e., such attempts have fallen far below thetheoretical yield). Typically, the enhancements were accomplished, bytransferring into host cells, genetic elements encoding enzymes thatdirect carbon flow into and/or through the common aromatic pathway. Suchgenetic elements can be in the form of extrachromosomal plasmids,cosmids, phages, or other replicons capable of transforming geneticelements into the host cell.

U.S. Pat. No. 5,168,056 to Frost described the use of a genetic elementcontaining an expression vector and a. gene coding for transketolase(Tkt), the tkt gene. This genetic element can be integrated into themicroorganisms chromosome to provide overexpression of the Tkt enzyme.

Additional examples include: Miller et al. (Miller, J. E., K. C.Backman, J. M. O'Connor, and T. R. Hatch, “Production of phenylalanineand organic acids by PEP carboxylase-deficient mutants of Escherichiacoli,”J. Ind. Microbiol., 1987, 2:143-149) who attempted to direct morecarbon flux into the amino acid pathway by use of a phosphoenolpyruvatecarboxylase (coded by ppc) deficient mutant; Draths et al. (Draths, K.M., D. L. Pompliano, D. L. Conley, J. W. Frost, A. Berry, G. L. Disbrow,R. J. Staversky, and J. C. Lievense, “Biocatalytic synthesis ofaromatics from D-glucose: The role of transketolase,” J. Am. Chem. Soc.,1992, 114:3956-3962) who reported that overexpression of transketolase(coded by tktA) and a feed-back resistant DAHP synthase (coded byaroG^(fbr)) resulted in improved production of DAHP from glucose.

The overproduction of transketolase in tkt transformed cells has beenfound to provide an increased flow of carbon resources into the commonaromatic pathway relative to carbon resource utilization in whole cellsthat do not harbor such genetic elements. However, the increased carbonflux may be further enhanced by additional manipulation of the hoststrain.

Thus, it is desirable to develop genetically engineered strains ofmicroorganisms that are capable of enhancing the production of DAHP tonear theoretical yield. Such genetically engineered strains can then beused for selective production of DAHP or in combination with otherincorporated genetic material for selective production of desiredmetabolites. Efficient and cost-effective biosynthetic production ofchorismate, quinic acid, hydroquinone, benzohydroquinone, catechol, orderivatives of these chemicals requires that carbon sources such asglucose, lactose, galactose, xylose, ribose, or other sugars beconverted to the desired product in high yields. Accordingly, it isvaluable from the standpoint of industrial biosynthetic production ofmetabolites to increase the influx of carbon sources for cellbiosynthesis of DAHP and its derivatives.

SUMMARY OF THE INVENTION

The present invention provides genetically engineered strains ofmicroorganisms that overexpress the pps gene for increasing theproduction of DAHP to near theoretical yields. The present inventionalso provides genetically engineered strains of microorganisms where atleast one of the plasmids pPS341, pPSL706, pPS706, or derivativesthereof is transformed into a microorganism for increasing theproduction of DAHP to near theoretical yields.

The present invention further provides a method for increasing carbonflow for the biosynthesis of DAHP in a host cell comprising the steps oftransforming into the host cell recombinant DNA comprising a pps gene sothat Pps is expressed at enhanced levels relative to wild type hostcells, concentrating the transformed cells through centrifugation,resuspending the cells in a minimal, nutrient lean medium, fermentingthe resuspended cells, and isolating DAHP from the medium.

The present invention further provides methods of increasing carbon flowinto the common aromatic pathway of a host cell comprising the step oftransforming the host cell with recombinant DNA comprising a pps gene sothat Pps is expressed at appropriate point in the metabolic pathways atenhanced levels relative to wild type host cells.

The present invention further provides methods for enhancing a hostcell's biosynthetic production of compounds derived from the commonaromatic pathway relative to wild type host cell's biosyntheticproduction of such compound, said method comprising the step ofincreasing expression in a host cell of a protein catalyzing theconversion of pyruvate to PEP.

The present invention also provides methods for overexpressing Pps inmicroorganism strains which utilize DAHP in the production DAHP ofmetabolites.

The present invention further provides a culture containing amicroorganism characterized by overexpressing Pps where the culture iscapable of producing DAHP metabolites near theoretical yields uponfermentation in an aqueous resuspension, minimal, nutrient lean mediumcontaining assimilable sources of carbon, nitrogen and inorganicsubstances.

The present invention further provides a genetic element comprising apps gene and one or more genes selected from the group consisting of anaroF gene, aroG gene, aroH gene, and an aroB gene.

The present invention further provides a DNA molecule comprising avector carrying a gene coding for Pps.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Overexpression of Pps increases the production of DAHP. The hoststrain used was AB2847 and the plasmids used are as labeled in thefigure. Note that pPSX1 and pUHE denote pPS341X1 and pUHE23-2,respectively. These strains were cultured first in the YE medium (a richmedium) to late stationary phase, and then washed and re-suspended in aminimal medium. (A) DAHP concentrations measured at 10 and 27 hoursafter re-suspension. (B) The activities of DAHP synthase (aroG) and Ppsmeasured at 27 hours after re-suspension.

FIG. 2. DAHP production at 10 and 27 hours after re-suspension. (A)Strain AB2847 with plasmids labeled in the abscissa. pPSX1 and pUHEdenote pPS341X1 and pUHE23-2, respectively. (B) Strains AB2847 (labeledas AB), JCL1283 ppc::Km (labeled as ppc), and JCL1362 pps::MudK (labeledas pps) with plasmids labeled in the abscissa.

FIG. 3. The reaction pathways for maximal conversion of glucose to DAHPfor (A) strains without Pps, (B) strains overexpressing Pps. The numbersare the relative fluxes needed for converting 7 moles of glucose toDAHP. The abbreviations are: G6P, glucose 6-P; F6P, fructose 6-P;1,6FDP, 1,6-fructose diphosphate; DHAP, dihydroxyacetone phosphate; GAP,glyceraldehyde 3-P, R5P, ribose 5-P; X5P, xylulose 5-P; S7P,sedoheptulose 7-P.

FIG. 4. The common aromatic pathway is shown whereby E4P and PEP undergo a condensation reaction to initiate the common aromatic pathway.

FIG. 5. The construction of pPSL706. Plasmids pPS706 and pGS103 wererestricted with EcoRI and ScaI. The fragments containing pps from pPS706and the fragment containing luxl′ were purified and ligated to generatepPSL706. Plasmid pPS706 was constructed by inserting a pps PCR fragmentinto 9 the cloning vector pJF118EH.

FIG. 6. Effects of Pps activity on DAHP production from glucose atdifferent IPTG and autoinducer concentrations. The strains areAB2847/pPSL706/pAT1 and AB2847/pPSL706/pRW5. Plasmid pGS 104 was used tosubstitute pPSL706 as a control, and the data are the leftmost point ineach graph.

DETAILED DESCRIPTION OF THE INVENTION

Many microorganisms synthesize aromatic precursors and aromaticcompounds from the condensation reaction of PEP and E4P to produce DAHP.This condensation reaction to form DAHP is the first committed step inthe biosynthetic pathway known as the common aromatic pathway. From thispathway, cells synthesize many cyclic metabolites, pre-aromaticmetabolites, and aromatic metabolites, such as the aromatic amino acids,quinone biomolecules, and related aromatic and cyclic molecules.

The inventor have found that cell lines can be developed that increasethe carbon flux into DAHP production and achieve near theoretical yieldsof DAHP by overexpressing phosphoenolpyruvate synthase (Pps) in the celllines. Overexpression of Pps can increase the final concentration andyield of DAHP by as much as two-fold, to a near theoretical maximum ascompared to wild type cell lines. The overexpression of Pps is achievedby transforming a cell line with recombinant DNA comprising a pps geneso that Pps is expressed at enhanced level relative to the wild typecell line and so that the yield of DAHP approaches its theoreticalvalue.

Generally, the present invention enhances expression in a host cell ofPps relative to a wild type host cell either by the transfer and stableincorporation of an extrachromosomal genetic element into the host cellor by the transfer of the genetic element into the genome of the hostcell. The expressed gene products are enzymes configured to provideappropriate catalytic sites for substrate conversion of common aromaticpathway compounds.

Besides the use of the pps gene, the present invention also provides fortransfer of genetic elements comprising the tkt gene, the gene codingfor DAHP synthase (aroF in E. coli), the gene coding for3-dehydroquinate synthase (arob in E. coli), or other genes encodingenzymes that catalyze reactions in the common aromatic pathway. Such acell transformation can be achieved by transferring one or more plasmidsthat contain genes that code for enzymes that increase the carbon fluxfor DAHP synthesis and for subsequent synthesis of other desired cyclic,pre-aromatic, and aromatic metabolites. As a result of this transfer ofgenetic element(s), more carbon enters and moves through the commonaromatic pathway relative to wild type host cells not containing thegenetic elements of the present invention.

In one embodiment, the present invention comprises a method forincreasing carbon flow into the common aromatic pathway of a host cellby increasing the production of DAHP through the overexpression of Ppsat the appropriate point in the common aromatic pathway to provideadditional PEP at the point where PEP condenses with E4P. Increasingcarbon flow requires the step of transforming the host cell withrecombinant DNA containing a pps gene so that Pps is overexpressed atenhanced levels relative to wild type host cells. DAHP is then producedby fermentation of the transformed cell in a nutrient medium where theDAHP can be extracted from the medium on a batch wise or continuousextraction procedure.

In another embodiment, the present invention involves theco-overexpression of a pps gene and other genes coding for enzymes ofthe common aromatic pathway where additional genetic material istransformed into the host cell. The genes transferred can include thetkt gene, DAHP synthase gene and DHQ synthase gene (preferably the aroFor aroB genes, respectively). Although the work so far has centeredaround transforming certain host cell strains of E. coli such as AB2847aroB, this particular host cell may not be the preferred host cells forthe commercial production of DAHP or DAHP metabolites through theoverexpression of Pps.

Another embodiment of the present invention is a method for enhancing ahost cell's biosynthetic production of compounds derived from the commonaromatic pathway. This method involves the step of increasing expressionof Pps in the host cell relative to a wild type host cell. The step ofincreasing expression of Pps can include transferring into the host cella vector carrying the pps gene. The overexpression of Pps results inforcing increased carbon flow into the biosynthesis of DAHP.

In another embodiment of the present invention, a method for enhancing ahost cell's biosynthetic production of compounds derived from the commonaromatic pathway relative to wild type host cell's biosyntheticproduction of such compound is provided. This method requires the stepof increasing expression in a host cell of a protein catalyzingconversion of pyruvate to PEP. The expression of such a protein caninvolve transferring into the host cell recombinant DNA including a ppsgene.

In another preferred embodiment, the present invention comprises agenetic element comprising the pps gene and a gene selected from thegroup consisting of a aroF gene, a aroB gene, and a tkt gene. Such agenetic element can comprise plasmid pPS341, a vector carrying a ppsgene.

To channel more carbon flux into the common aromatic pathway, theinventor has found that PEP production in a given cell line must beincreased. This increase can be achieved by deactivating pathwayscompeting for PEP or by recycling pyruvate back into PEP.

Besides being used in the biosynthesis of DAHP, PEP is used as aphosphate donor in the phosphotransferase system (PTS) which isresponsible for glucose uptake. Additionally, PEP can be converted topyruvate by pyruvate kinases and to oxaloacetate by phosphoenolpyruvatecarboxylase. All such competing pathways limit the availability of PEPfor the biosynthesis of DAHP and all metabolites derived from the commonaromatic pathway or pathways branching therefrom.

Once PEP is converted to pyruvate by either PTS or pyruvate kinases,pyruvate is not generally recycled back to PEP because of a high energycost. As a result, a large amount of carbon flux is channeled from PEPthrough pyruvate and eventually to organic acids, carbon dioxide, orcell mass.

PEP is critical for the biosynthesis of DAHP and DAHP metabolitesincluding all metabolites from the common aromatic pathway. The firstcommitted step of the common aromatic pathway involves the formation ofDAHP from the condensation of E4P and PEP. This condensation involves analdol condensation between an intermediate carbanion of C-3 of PEP andthe carbonyl C-1 of E4P. The majority of the PEP molecules reactstereospecifically with respect to the configuration on C-3.

A key component of the methods of the present invention directed atincreased carbon flux commitment to DAHP and DAHP metabolites is therecycling of pyruvate to PEP. Pyruvate is available in host cells as anend product of glycolysis.

In glycolysis, the free energy of degradation of glucose to pyruvate isutilized to synthesize ATP. Broadly speaking, this process involves aninvestment of ATP to form a phosphoryl compound (FBP) from glucose,which is then cleaved to two C₃ units. The free energy of this reactionis used in the oxidation of GAP which is then utilized to synthesize anacyl phosphate, a “high-energy” intermediate (1,3-BPG). 1,3-BPG is usedto phosphorylate ADP to ATP. The second “high-energy” compound of thepathway, PEP, which is produced from 2PG, also phosphorylates ADP toATP. Thus, the degradation of glucose via the glycolytic pathwayproduces pyruvate. The overall reaction of glycolysis is therefore:

Glucose+2ADP+2P+2NAD+>2 pyruvate+2ATP+2NADH+4H+2H20

The first step of glycolysis is the transfer of a phosphoryl group fromATP to glucose to form glucose-6-phosphate (G6P) in a reaction catalyzedby hexokinase. Hexokinase is a relatively nonspecific enzyme containedin all cells that catalyzes the phosphorylation of hexoses such asD-glucose, D-mannose, and D-fructose. The second substrate forhexokinase, as with other kinases, is an Mg²⁺—ATP complex. In fact,uncomplexed ATP is a potent competitive inhibitor of hexokinase.

Hexokinase has a Random Bi Bi mechanism in which the enzyme forms aternary complex with glucose and Mg²⁺—ATP before the reaction occurs. Bycomplexing with the phosphate oxygen atoms, the Mg²⁺ is thought toshield their negative charges, making the phosphorus atom moreaccessible for the nucleophilic attack of the C(6)—OH group of glucose.

Next G6P is converted to fructose 6-phosphate (F6P) by phosphoglucoseisomerase (PGI). This reaction is an isomerization of an aldose to aketose. Since G6P and F6P both exist predominantly in their cyclicforms, the reaction requires ring opening, followed by isomerization,and subsequent ring closure. The overall reaction is thought to occur byenzyme mediated general acid-base catalysis.

Next, phosphofructokinase (PFK) phosphorylates F6P to yield fructose1,6-bisphosphate (FBP or F1,6P). PFK catalyzes the nucleophilic attackby the C(1)—OH group of F6P on the electrophilic y-phosphorus atom ofthe Mg²⁺—ATP complex.

PFK plays a central role in control of glycolysis because it catalyzesone of the pathway's rate-determining reactions. In many organisms theactivity of PFK is enhanced allosterically by several substances,including AMP, and inhibited allosterically by several other substances,including ATP and citrate. The regulatory properties of PFK areexquisitely complex.

Aldolase then catalyzes the cleavage of FBP to form the two triosesglyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).This reaction is an aldol cleavage (the reverse of an aldolcondensation). The aldol cleavage between. C(3) and C(4) of FBP requiresa carbonyl at C(2) and hydroxyl at C(4).

Only one of the products of the aldol cleavage reaction, GAP, continuesalong the glycolytic pathway. However, DHAP and GAP are ketose-aldoseisomers just as are F6P and G6P. Interconversion of GAP and DHAP istherefore possible via an enediolate intermediate in analogous to thephosphoglucomutase reaction. Triose phosphate isomerase (TIM) catalyzesthis process.

At this point, the glucose which has been transformed into two GAPs, hascompleted the preparatory stage of glycolysis. This process has requiredthe expenditure of two ATPs. However, this investment has resulted inthe conversion of one glucose into two C₃ units, each of which has aphosphoryl group that, with a little chemical artistry, can be convertedto a “high-energy” compound whose free energy of hydrolysis can becoupled to ATP synthesis. This energy investment will be doubly repaidin the final stage of glycolysis in which the two phosphorylated C₃units are transformed to two pyruvates with the coupled synthesis offour ATPs per glucose.

The next step in glycolysis involves the oxidation and phosphorylationof GAP by NAD⁺ and P, as catalyzed by glyceraldehyde 3-phosphatedehydrogenase (GAPDH). In this reaction, aldehyde oxidation, anexergonic reaction, drives the synthesis of the acryl phosphate1,3-diphosphyglycerate (1,3-BPG).

The next reaction of the glycolytic pathway results in the firstformation of ATP together with 3-phosphoglycerate (3PG) in a reactioncatalyzed by phosphoglycerate kinase (PGK):

3PG is then converted to 2-phosphoglycerate (2PG) by phosphoglyceratemutase (PGM). This reaction is necessary preparation for the nextreaction in glycolysis, which generates a “high-energy” phosphorylcompound for use in ATP synthesis.

Subsequently, 2PG is dehydrated to phosphoenolpyruvate (PEP) in areaction catalyzed by enolase. The enzyme forms a complex with adivalent cation such as Mg²⁺ before the substrate is bound. Fluoride ioninhibits glycolysis with the accumulation of 2PG and 3PG. It does so bystrongly inhibiting enolase in the presence of P_(i). The inhibitoryspecies is fluorophosphate ion (FPO₃ ³⁻), which probably complexes theenzyme-bound Mg²⁺ thereby inactivating the enzyme. Enolase's substrate2PG, therefore builds up and as it does so, is equilibrated with 3PG byPGM.

Finally, pyruvate kinase (PK) couples the free energy of PEP hydrolysisto the synthesis of ATP to form pyruvate. At this point, glycolysis hasproduced PEP, one of the precursors to DAHP production and the entry wayto the common aromatic pathway.

Besides PEP, DAHP biosynthesis, as well as that of other productsderived from the common aromatic pathway and pathways branchingtherefrom, depends on the biosynthesis of E4P.

With reference to FIG. 4, E4P is a biosynthetic intermediate of thepentose phosphate pathway. The pentose phosphate pathway is situatedbetween glycolysis and a variety of different biosynthetic cascades.This pathway yields E4P via a nonoxidative branch of the pathway. Thenonoxidative pentose phosphate pathway converts D-fructose 6-phosphateinto varying equivalents of D-ribose 5-phosphate, D-sedoheptulose7-phosphate, and E4P. The first two end products are associated with thebiosynthesis of nucleotides and gram-negative bacteriallipopolysaccharides, respectively, while E4P is a precursor to thearomatic amino acids: phenylalanine, tyrosine, and tryptophan.

The initial siphoning of intermediates from glycolysis by the pentosephosphate pathway involves transketolase catalyzed transfer of a ketolgroup from D-fructose 6-phosphate to D-glyceraldehyde 3-phosphate toform E4P and D-xylulose 5-phosphate. Pentose phosphate epimerase thenconverts the D-xylulose 5-phosphate into D-ribulose 5-phosphate followedby pentose phosphate isomerase mediated transformation of the D-ribulose5-phosphate into D-ribose 5-phosphate.

At this stage, the D-ribose 5-phosphate can be exploited bytransketolase as an acceptor of a ketol group derived from anothermolecule of D-fructose 6-phosphate forming a second molecule of E4P andD-sedoheptulose 7-phosphate. Finally, the enzyme transaldolase catalyzestransfer of a dihydroxyacetone group from the D-sedoheptulose7-phosphate to D-glyceraldehyde 3-phosphate yielding the third moleculeof E4P and D-fructose 6-phosphate. Thus, the nonoxidative pentosephosphate pathway achieves net conversion of two molecules of D-fructose6-phosphate into three molecules of E4P.

The condensation of PEP and E4P is catalyzed by the enzyme DAHPsynthase. Many microorganisms, including E. coli, produce three DAHPsynthase isoenzymes: phenylalanine-sensitive DAHP synthase (phe),tyrosine-sensitive DAHP synthase (tyr), and tryptophan-sensitive DAHPsynthase (trp). The tetrameric DAHP synthase (phe) has a subunitmolecular weight of 35,000. The dimeric DAHP synthase (tyr) and DAHPsynthase (trp) have subunit molecular weights approaching 40,000. Thenative forms of the enzymes are probably protein-PEP adducts. In E. colithe structural genes for DAHP synthase (tyr), DAHP synthase (phe), andDAHP synthase (trp) are aroF, aroG, and aroH, respectively. These genesare located at 56, 17, and 37 min, respectively, on the E. coli linkagemap.

In wild-type E. coli, 80% of the total DAHP synthase activity iscontributed by the phenylalanine-sensitive isoenzyme, while 20% iscontributed by the tyrosine-sensitive isoenzyme. There are only tracesof the DAHP synthase (trp) in E. coli.

After the condensation of PEP and E4P, the next reaction of the commonaromatic pathway is an intramolecular exchange of the DAHP ring oxygenwith C-7, accompanied by an oxidation at C-6 and a reduction at C-2.Cleavage of the phosphoester provides the driving force to form3-dehydroquinate (DAH). This reaction is catalyzed by dehydroquinatesynthase (DAH synthase).

Pure DAH synthase is a single polypeptide chain having a molecularweight of 40,000-44,000. The enzyme requires Co and NAD for activity,the latter in catalytic amounts. The formation of 3-dehydroquinate fromDAHP is stereospecific and proceeds with inversion at the C-7 of DAHPwithout exchange of hydrogen with the growth medium.

Quinoid organics are formed from pathways which branch from the commonaromatic and utilize DAH. A stereospecific syn-dehydration of3-dehydroquinate introduces the first double bond of the aromatic ringsystem to yield 3-dehydroshikimate. The reaction is catalyzed by3-dehydroquinate dehydratase. Schiff base formation between enzyme andsubstrate causes a conformational change in the substrate (twisted boat)that leads to the stereospecific course of the reaction.

Shikimate biosynthesis from 3-dehydroshikimate is catalyzed by shikimatedehydrogenase. This NADP-specific enzyme facilitates the hydrogentransfer from the A-side of NADPH.

Shikimate is phosphorylated to shikimate 3-phosphate by shikimatekinase. Shikimate kinase is a polypeptide of 10,000 daltons that iscomplexed with the bifunctional DAHP synthase-chorismate mutase. Thekinase, only active in the complex, has been purified to homogeneity.Since the enzyme is inhibited by chorismate, prephenate, ADP, and5-enolpyruvoylshikimate 3-phosphate are derepressed by growth onlimiting tyrosine, shikimate kinase is believed to represent a keyallosteric control point of the pathway in some types of host cells.

Shikimate-3-phosphate reacts with PEP to form 5-enolpyruvoyl shikimate3-phosphate and inorganic phosphate. The reversible enzyme-catalyzedreaction is a transfer of an unchanged enolpyruvoyl moiety of PEP.Protonation of C-3 of PEP combined with a nucleophilic attack of the5-hydroxyl of shikimate leads to a presumed intermediate from which5-enolpyruvoylshikimate 3-phosphate is obtained in a 1,2-elimination oforthophosphate. The reaction is catalyzed by 5-enolpyruvoylshikimate3-phosphate synthase.

The second double bond in the aromatic ring system is introduced througha trans-1,4-elimination of orthophosphate from 5-enolpyruvoyl shikimate3-phosphate to yield chorismate. The reaction is catalyzed by chorismatesynthase.

From chorismate, the endpoint of the common aromatic pathway,biosynthesis of a diverse number of aromatic compounds is possible. Forexample, as indicated in FIG. 4, the aromatic amino acids tryptophan,tyrosine, and phenylalanine can be synthesized from chorismate alongtheir respective biosynthetic pathways. As previously noted, othercommercially important aromatic compounds also produced from chorismateinclude folates, aspartame, melanin, prephenic acid, and indigo.

In addition to the common aromatic pathway, other pathways utilizingDAHP produce other aromatic metabolites. For example, catechol andquinoid organics, such as quinic acid, benzoquinone and hydroquinone,can be produced from pathways branching from the common aromaticpathway.

According to theoretical analyses, the inventor believes that themaximum yield of DAHP and aromatic amino acids from glucose can beincreased by two-fold if pyruvate is recycled back to PEP. The maximumyield may be calculated by assuming that the branched pathways areblocked and that the carbon flow is directed by the most efficientpathways with minimum loss to carbon dioxide and other metabolites.Under these conditions and under steady state conditions, the relativeflux through each step can be calculated by balancing the input andoutput fluxes from each metabolite pool.

As shown in FIG. 3A, for maximum yield of DAHP production by strainswithout Pps overproduction, 7 moles of glucose are needed to produce 3moles of DAHP (43% molar yield) and 7 moles of pyruvate which is furthermetabolized. The relative flux through each intermediate step is alsoshown in FIG. 3A. The formation of pyruvate is necessary because of thestoichiometry of the phosphotransferase system for glucose uptake.

In the presence of glucose, pyruvate is not recycled back to PEPefficiently because the enzyme Pps is not induced. The inventor hasfound that pyruvate is effectively recycled to PEP via overexpressedPps, even in the presence of glucose, resulting in a two-fold, increasein DAPH which approaches, if not achieves, the theoretical levels forDAHP synthesis.

As shown in FIG. 3B, at the theoretical maximum, 6 moles of DAHP can beproduced from 7 moles of glucose (86% molar yield). The nonoxidativepart of the pentose pathway provides E4P, while overexpression of Ppsrecycles pyruvate back to PEP.

The data described above and shown in FIGS. 3A and 3B are in agreementwith this flux distribution model. Controls with inactive Pps and withno Pps demonstrate that enhanced activity, through overexpression ofPps, is required to achieve high yields of DAHP and, of course, DAHPmetabolites including metabolites of the common aromatic pathway.

In previous work, the inventor demonstrated that overexpression of Ppsin host cells cultured on nutrient rich, glucose containing medium ledto growth inhibition, increased glucose consumption, and excretion ofpyruvate and acetate. Their previous study also showed that the effectsof Pps overexpression on DAHP production, in actively growing cultures,are not as significant, and that the adverse effects of Ppsoverexpression on cell growth negated any beneficial effects on DAHPproduction.

The stimulation of glucose consumption in the previous work wasattributed to the altered PEP/pyruvate ratio. It was hypothesized thatincreased PEP/pyruvate ratio stimulates the phosphotransferase systemfor increased glucose consumption, which in turn results in theexcretion of pyruvate.

The inventor discovered that the problem of growth impairment could beovercome through the use of high density re-suspension cultures grown innutrient lean, glucose media. Such re-suspension cultures attain highmetabolic activity with low growth rates. This discovery led to DAHPyields approaching theoretical values.

In the present invention, PEP was redirected to the aromatic pathway,and thus the PEP/pyruvate ratio was decreased. This flux redirectionexplains the insensitivity of the specific glucose consumption rate toPps overexpression in the experimental system of the present invention.The increased DAHP production from glucose caused by Pps overexpressionalso suggests that Pps actually functions in its physiological direction(from Pyruvate to PEP ) in vivo, even under glycolytic conditions.

PEP is also a precursor to the pathways that utilize the Ppc enzymecoded by the ppc gene. It has been reported that the deletion of ppcincreased the production of phenylalanine and acetate. Moreover, it hasbeen shown that the overexpression of Ppc in a wild-type host reducesacetate production. Both results may indicate that the flux through Ppc(from PEP to OAA) is reasonably significant under those conditions, andthus, the modulation of Ppc expression level may affect the utilizationof PEP. However, in the present invention, deleting the chromosomal ppcgene did not have a positive effect on DAHP production, suggesting thatthe flux through Ppc is not important in the methods of the presentinvention.

One preferred embodiment of the present invention encompassesmodification of a host cell to cause overexpression of an enzyme havingthe catalytic properties of naturally derived Pps, and, therebymaximizing the yield of DAHP to near theoretical yields. Enzymes havingthe catalytic activity of Pps include, but are not limited to, Ppsproduced by expression in whole cells of a naturally derived pps gene,enzymes produced by expression in whole cells of a naturally derived ppsgene modified by sequence deletion or addition so that the expressedenzyme has an amino acid sequence that varies from unmodified Pps,abzymes produced to have catalytic sites with steric and electronicproperties corresponding to catalytic sites of Pps, or other proteinsproduced to have the capability of catalyzing the conversion of pyruvateto PEP by any other art recognized means.

In another preferred embodiment, the inventor has observed that the Ppseffect on DAHP production is enhanced by the simultaneous overexpressionof Tkt. Such simultaneous overexpression ensures that both precursorsnecessary for DAHP biosynthesis are overproduced. Although simultaneousoverexpression of Pps and Tkt may be required to attain near theoreticalyields of DAHP, the overexpression of Pps alone in the methods of thepresent invention significantly enhanced DAHP production over Tktoverexpression alone (FIG. 2A). This result may suggest that withoutpyruvate recycling mediated by Pps (FIG. 3A), sufficient PEP flux toDAHP synthesis cannot be achieved.

Additionally, the transformation of DNA, including the pps gene, intomicroorganisms engineered for the overexpression of other substrates,and/or overexpression or derepression of enzymes in the pentosephosphate or common aromatic pathway can be used to tailor themicroorganism to achieve near theoretical yields of such DAHPmetabolites as tyrosine, tryptophan, phenylalanine, and other aromaticmetabolites such as indigo, catechol and quinoid organics such as quinicacid, benzoquinone, and hydroquinone.

Enzymes catalyzing reactions in the pentose phosphate or common aromaticpathway include those enzymes produced by expression in whole cells ofnaturally derived pentose phosphate or common aromatic pathway genes,enzymes produced by expression of naturally derived pentose phosphate orcommon aromatic pathway genes that have been modified by sequencedeletion or addition so that the expressed enzyme has an amino acidsequence that. differs from the natural enzyme, or abzymes havingcatalytic sites with steric and electronic properties corresponding tocatalytic sites of a natural enzyme in the common aromatic or pentosephosphate pathway.

Pps or enzymes having Pps-like catalytic activity can be overexpressedrelative to Pps production in wild type cells (as measured by standardPEP synthase activity assays known in the art and described inExample 1) in conjunction with any number of other enzymes in the commonaromatic pathway or pathways branching therefrom. For example,overexpression of Pps, DAHP synthase, and transketolase; Pps, DHQsynthase, and transketolase; Pps, DAHP synthase, DHQ synthase, andtransketolase; Pps, transketolase, and shikimate kinase; Pps,transketolase (Tkt), and chorismate mutase; or any other common aromaticpathway enzymes in conjunction with Pps overproduction can enhancecarbon source input to and/or throughput of the common aromatic pathway.

Enhanced expression of genes coding for proteins able to perform orcontrol pentose phosphate or common aromatic pathway enzymatic functionsis mediated by genetic elements transferable into a host cell. Geneticelements as herein defined include nucleic acids (generally DNA or RNA)having expressible coding sequences for products such as proteins,apoproteins, or antisense RNA, which can perform or control pentosephosphate or common aromatic pathway enzymatic functions. The expressedproteins can function as enzymes, as repressor or derepressor agents, orto control enzyme expression.

The nucleic acids coding these expressible sequences can be eitherchromosomal (e.g., integrated into a host cell chromosome by homologousrecombination) or extrachromosomal (e.g., carried by plasinids, cosmids,etc.). In addition, genetic elements are defined to include optionalexpression control sequences including promoters, repressors, andenhancers that act to control expression or derepression of codingsequences for proteins, apoproteins, or antisense RNA. For example, suchcontrol sequences can be inserted into wild type host cells to promoteoverexpression of selected enzymes already encoded in the host cellgenome, or alternatively can be used to control synthesis ofextrachromosomally encoded enzymes.

The genetic elements of the present invention can be introduced into ahost cell by a genetic agent including, but not limited to, plasmids,cosmids, phages, Yeast artificial chromosomes or other vectors thatmediate transfer of the genetic elements into a host cell, or mixturesthereof. These vectors can include an origin of replication along withcis-acting control elements that control replication of the vector andthe genetic elements carried by the vector. Selectable markers can bepresent on the vector to aid in the identification of host cells intowhich the genetic elements have been introduced. For example, selectablemarkers can be genes that confer resistance to particular antibioticssuch as tetracycline, ampicillin, chloramphenicol, kanamycin, orneomycin.

A preferred means for introducing genetic elements into a host cellutilizes an extrachromosomal multi-copy plasmid vector into whichgenetic elements in accordance with the present invention have beeninserted. Plasmid borne introduction of the genetic element into hostcells involves an initial cleaving of a plasmid with a restrictionenzyme, followed by ligation of the plasmid and genetic elements inaccordance with the invention. Upon recircularization of the ligatedrecombinant plasmid, transformation or other mechanisms for plasmidtransfer (e.g., electroporation, microinjection, etc.) is utilized totransfer the plasmid into the host cell.

Plasmids suitable for insertion of genetic elements into the host cellinclude, but are not limited to, pBR322 and its derivatives such aspAT153, pXf3, pBR325, and PBR327, pUC vectors, pACYC and itsderivatives, pSC101 and its derivatives, and ColEI. U.S. Pat. No.5,168,056, incorporated herein by reference, teaches the incorporationof the tkt gene which codes for the enzyme Tkt into host cell. Tktcatalyzes the conversion of the carbon source D-fructose-6-phosphate toE4P, one DAHP precursor.

Suitable host cells for use in the present invention are members ofthose genera capable of being utilized for industrial biosyntheticproduction of desired aromatic compounds. Accordingly, host cells caninclude prokaryotes belonging to the genera Escherichia,Corynebacterium, Brevibacterium, Arthrobacter, Bacillus, Pseudoinonas,Streptomyces, Staphylococcus, or Serratia. Eukaryotic host cells canalso be utilized, with yeasts of the genus Saccharoinyces orSchizosaccharoinyces being preferred.

More specifically, prokaryotic host cells suitable for use in thepresent invention include, but are not limited to, Escherichia coli,Corynebacterium glutamicuni, Corynebacterium herculis, Brevibacteriunidivaricatum, Brevibacterium lactofermeiztuimi, Brevibacterium flavum,Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacilluscoagulans, Bacillus lichenfonis, Bacillus megaterium, Bacillusmesenztericus, Bacillus pumilis, Bacillus subtilis, Pseudoliioizasaerugiizosa, Pseudomonas aiigulata, Pseudomnonas fluorescent,Pseudoiizoizas tabaci, Streptoomyces aureofaciens, Streptonzycesavernzitilis, Streptomyces coelicolor, Streptonzyces griseus,Streptolnyces kasugensis, Streptoiyces lavendulae, Streptomyceslipnianii, Streptornyces lividans, Staphylococcus epidermis,Staphylococcus saprophyticus, or Serratia marcescens and theirgenetically engineered strains or mixtures thereof. Preferred eukaryotichost cells include Saccharoniyces cerevisiae or Saccharomycescarlsbergensis and their genetically engineered strains or mixturesthereof.

For industrial production of primary metabolites derived from chorismate(such as aromatic amino acids), deregulated mutant strains of the aboverecited species that lack feedback inhibition of one or more enzymes inthe metabolic biosynthetic pathway are preferred. Such strains can becreated by random or directed mutagenesis, or are commerciallyavailable. Examples of E. coli strains having DAHP synthase, prephenatedehydratase, or chorismate mutase feedback inhibition removed aredescribed in U.S. Pat. No. 4,681,852 to Tribe and U.S. Pat. No.4,753,883 to Backman et al., incorporated herein by reference.

To overcome the stoichiometric limitations in the condensation of E4Pand PEP, the present invention overexpresses Pps in the presence ofglucose and directs more carbon flux into the production of DAHP.

The following list of abbreviations for compounds commonly noted in thespecification and Examples is presented as follows:

DHQ 3-dehydroquinate DAH 3-deoxy-D-arabino-heptulosonic acid DAHP3-deoxy-D-arabino-heptulosonic acid 7-phosphate TSP3-(trimethylsilyl)propionic-2, 2, 3, 3-d sub 4 acid, sodium salt PEPPhosphoenol pyruvate NADH beta-nicotinamide adenine dinucleotidephosphate, reduced form Kan kanamycin Ap ampicillin Tc tetracycline Cmchloramphenicol

Strains and Plasmids

Escherichia coli AB2847 aroB mal T6^(r), obtained from E. coli GeneticStock Center, Yale University, was used as the preferred host strain forDAHP production. BJ502 tkt-2 fhuA22 garB10 ompF627 fadL701 relA1 pit-10spoT1 mcrB1 phoM510, also from E. coli Genetic Stock Center, was used inthe identification of the tkt clone. JCL1242 ppc::Km was constructed asdescribed previously by inserting a kanamycin cassette into a cloned ppcgene, which is then integrated into the chromosome by homologousrecombination.

Plasmid pPS341 was constructed by cloning a fragment of E. colichromosomal DNA containing pps gene into an IPTG-inducible expressionvector pUHE23-2 (a pBR322 derivative) as taught by Patnaik et al., andthe contents of which are herein incorporated by reference. PlasmidpPS341X1 containing the inactive gene product of pps was constructed bycodon insertion mutagenesis, the details of wvlhich are fully describedin Patnaik et al. The pps gene on pPS341 was inserted with a Mu dII1734lac⁺ Km^(r) (MudK) according to published protocol of Castiho et al.,the contents of which are herein incorporated by reference. Briefly, aMu lysate was made from a donor strain POII1734/pPS341, which waslysogenized by the mini-Mu element and a Mu cts. The lysate was used toinfect a Mu lysogen of HG4 pps pck, and colonies were selected forAp^(r) and Km^(r) simultaneously to ensure that the mini-Mu elementhopped to the plasmid. Colonies were further screened for Pps⁻ phenotype(inability to grow on pyruvate). Restriction analysis of plasmid DNAfurther confirmed the insertion of the MudK element into the pps gene onplasmid pPS341. 20% of these selected colonies showed IPTG-dependentexpression of β-galactosidase, indicating an in-frame insertion. Plasmidfrom one such colony was named pPS1734, which was then linearized at theScal site, and then transformed into strain JC7623 recB21 recC22 sbcB15.Transformants were selected for Km^(r) and scored for Ap sensitivity.Such colonies presumably contained pps::MudK on the chromosome. By useof P1 transduction, this locus was moved to AB2847 and Km^(r)transductants were further screened for inability to grow on pyruvate.One such colony was designated JCL1362 and used for later studies. TheMudK insertion into chromosomal pps was further confirmed bycotransduction frequency (89%) with Tet^(r) marker from strain CAG12151zdh-925::Tn10.

Plasmid pRW5, Genencor International, South San Francisco, Calif., is apACYC derivative and contains aroG^(fbr). This plasmid also contains alad gene, and the aroGfbr is expressed from a lac promoter. To constructplasmid pAT1 containing both aroG^(fbr) and tktA, a 5-Kb BamH1 fragmentof E. coli DNA was cut from page 473 of the Kohara miniset (NationalInstitute of Genetics, Japan), and was inserted into the BamH1 site ofpRW5. This fragment was reported to contain the tktA gene, and wasconfirmed by its ability to complement a tkt strain (BJ502) for growthon ribose, and also from the migration distance of the gene product asjudged on a 12% SDS-PAGE (molecular weight ca. 72,500).

Construction of pPS706 and the Control

The plasmid pPS706 was constructed by inserting a 2.4 kb PCR fragmentcontaining the promoter-less pps gene into the vector pJF118EH. Theprimers were designed from the published pps sequence and contained anEcoRI site and a φ10 ribosome binding site upstream of the pps sequenceand a BainzHl site downstream of the sequence. The PCR product was thencloned into the EcoRI and BaZizHI sites of pJF118EH. Positive cloneswere selected based on complementation of HG4 pps for growth onpyruvate. Expression of pps from this construct is controlled by the tacpromoter inducible by IPTG.

The plasmid pPSL706 was then constructed from pPS706 as shown in FIG. 5.Briefly, a ScaI/EcoRI fragment containing the pps gene was cut frompPS706 and purified from the restriction buffer. This fragment was thencloned into a purified ScaI-EcoRI fragment containing the luxI′ promoterfrom pGS103, kindly given to the inventor by Tom Baldwin. Department ofBiochemistry and Biophysics, Texas A&M University. Expression using thissystem is controlled by the autoinducer (AI) in the culture media.pPSL706 is ampicillin resistant and compatible with other pACYC184derivatives such as pRWS and pATI. The strains and plasmids used aresummarized in Table I and Table II.

TABLE I Bacterial Strains Strain Relevant genotype Source AB2847 aroBmal T6^(r) Genetic Stock Center BJ502 tkt-2 Genetic Stock Center JC7623recB21 recC22 sbcB15 (26) HG4 pck-2 pps-3 Hughes Goldie POII1734 araD139ara::(Mu cts)3 Δ(lac)X74 (4) gal U galK rpsL with Mu dII1734lac⁺(Km^(r)) CAG12151 zdh-925::Tn10 (22) JCL1242 VJS676 but ppc::Km (5)JCL1283 AB2847 but ppc::Km This study JCL1362 AB2847 but pps::Mu dII1734This study

TABLE II Plasmids Plasmids Relevant genotype Source pUHE23-2 Ap^(r);IPTG-inducible T7(A1) H. Bujard early promoter pPS341 same as pUHE23-2but pps⁺ (18) pPS341X1 same as pPS341 but pps-50 (18) (2-codoninsertion) pPS1734 pPS341::Mu dII1734 (Km^(r)lac⁺) This study pRW5pACYC184 derivative Cm^(r), but Genencor International tandem lacpromoters aroG^(fbr+) pAT1 same as pRW5 but tktA⁺ This study pPS706 aspJF118EH but pps⁺ This study pPSL706 as pGS104 but pps⁺ This study

Media and Growth Conditions

All cloning procedures were carried out in Luria-Bertani medium. YEmedium contained K₂HPO₄ (14 g/L), KH₂PO₄ (16 g/L), (NH₄)₂SO₄ (5 g/L),MgSO₄ (1 g/L), yeast extract (15 g/L), and D-glucose (15 gIL). Minimalmedium used for high-cell density re-suspension cultures contains perliter, K₂HPO₄ (14 g), KH₂PO₄ (16 g), (NH₄)₂SO₄ (5 g), MgSO₄ (1 g),D-glucose (15 g) and was also supplemented with thiamine (1 mg),shikimic acid (50 mg), L-tyrosine (8 mg), L-phenylalanine (8 mg), andL-tryptophan (4 mg). The minimal medium was supplemented with succinate(0.1 g/L) when growing the ppc mutant and its control. For stablemaintenance of plasmids, ampicillin (100 mg/ml), chloramphenicol (50mg/ml) were added to the culture medium. Concentration of theantibiotics were reduced by half when minimal medium was used.

Overnight cultures in YE medium were grown at 37° C. in a roller drumand then were subcultured in the same medium with appropriateenablement. Cultures were grown in 250 ml shake flasks at 37° C. in agyratory water bath shaken at 200 rpm. After four hours of incubationOD₅₅₀: 2-3) cultures were induced withisopropyl-β-D-thiogalactopyranoside, IPTG (1 mM). Cells were harvestedfrom late stationary phase by centrifugation at 6000 ×G and were washedtwice with minimal medium before re-suspending in the same minimalmedium supplemented with appropriate enablement and IPTG (1 mM). InitialOD₅₅₀ of all high-density re-suspension cultures were about 4.0. Theoptical density can be greater than 4.0. In other terms, the media maycontain at least 5×10⁹ cells/mL. Samples from the re-suspension cultureswere withdrawn periodically for assaying DAH(P) and glucoseconcentration in the medium.

Determination of Glucose and DAHP

Cells were removed from samples by centrifugation and the supernatantswere stored at 4° C. until all samples had been collected. Residualglucose in the culture supernatant was determined by thedinitrosalicylic acid assay for total reducing sugars. For additionalinformation an this assay, see Miller (Miller, G. L. 1958. Use ofdinitrosalicylic acid reagent for determination of reducing sugars.Anal. Chem. 31;426-428) and Patnaik et al. ( Patnaik, R., W. D. Roof, R.F. Young, and J. C. Liao. 1992. Stimulation of glucose catabolism inEscherichia coli by a potential futile cycle. J. Bacteriol.174:7527-7532), the contents of which are herein incorporated byreference. The concentration of DAH(P) in the supernatant was determinedby the thiobarbiturate assay. For additional information an thin assay,see Draths et al. and Gollub et al. (Gollub, E., H. Zalkin, and D. B.Sprinson. 1971. Assay for 3-Deoxy D-arabino-heptulosonic Acid7-phosphate Synthase. Methods in Enzymology 17A:349-350), the contentsof which are herein incorporated by reference. This assay does notdistinguish between DAH and DAHP.

Enzyme Assays

Cells were harvested by centrifugation at 6000×G and were washed andre-suspended in potassium phosphate buffer (50 mM) pH 7 or 5 mMTris-Cl-1 mM MgCl₂ (pH 7.4), for DAHP, synthase or PEP synthase (Pps)assay, respectively. Cell extracts were prepared by rupturing cellsthrough a French pressure cell (SLM Aminco, Urbana, Ill.) at 160,000lb/in² DAHP synthase activity was assayed by the procedure of Schoner asdescribed more fully in Schoner et al. (Schoner, R. and K. M. Herrmann.1976. 3-Deoxy-D-arabino-heptulosonate 7-phosphate Synthase. J. Biol.Chem. 251:5440-5447), the contents of which are herein incorporated byreference. Pps activity was assayed as described previously. Totalprotein in the extracts was determined with the Bio-Rad dye reagent(Bradford assay) with bovine serum albumin as the standard.

Effects on Pps on DAHP Production from Glucose

The purpose of constructing pPS706 was to express Pps with an induciblepromoter not affected by IPTG. This plasmid, together with pRW5,provided a means to vary the activities of the enzymes, Pps and AroG,independently Under the control of two different promoters. The thirdenzyme, TktA, was under the. control of its natural promoter and thusvariable in only an on/off mode (presence or absence of the gene). Thissystem then allowed the examination of Pps effect over a wider range ofconditions. Moreover, it is possible that this system would show anoptimal point where the enzyme activities were high enough to providemaximum production of DAHP but not so high as to exert a protein load onthe system decreasing DAHP production as a result. The inventortherefore measured DAHP production by AB2847/pRW5/pPS706 andAB2847/pATI/pPS706 in a glucose medium at varying IPTG and autoinducer(N-(3-xox-hexanoyl)-hmoserine lactone) concentrations.

FIG. 6 shows the efffect of Pps at various AI and IPTG concentrations inglucose medium. At low IPTG concentrations (low AroG activities), Ppshas little or no effect. When IPTG concentration exceeded 50 mM, Ppseffect began to show. Plasmid pGS104, isogenic with pPS706 except forthe pps locus, was used as a control, and it showed no effect with orwithout the addition of AI. The Pps effect was more significant in thestrain overexpressing TktA, and it was not due to a variation in AroG orTktA activities, since AroG and TktA activities were shown to beconstant with or without Pps overexpression. From the measurement ofresidual glucose (data not shown), the yields of DAHP from glucosereached 100%, which corresponds to 70-80% after adjusting for theoverestimation of DAHP. The latter value is. consistent with thatpredicted by the stoichiometric analysis, which indicates a maximumtheoretical yield of 86% from glucose when pyruvate is recycled to PEPby Pps. Although increases in DAHP levels and yields with Pps activitywere observed, a drop with higher Pps activity which would have provideda peak was not evident. The levels of DAHP instead seem to reachsaturation with further induction of Pps.

Formation of byproducts

To gain insight into the metabolic flux distribution, the culture brothwas analyzed for fermentation byproducts by use of HPLC. Samples weretaken from cultures in glucose media with varying activities of Pps,AroG, and TktA. Results indicate that the host strain AB2847 producedacetate, succinate, and formate as the major byproducts when neitherAroG nor Pps was overexpressed. Production of these acids generallydecreased with the increase in IPTG concentration, except formate. Thisdecrease correlates with the increase in DAHP production. WhenAB2847/pAT1/pPS706 was cultured in glucose with IPTG concentrationbeyond 50 mM, the broth had undetectable levels of these acids (data notshown). While levels of formic and acetic acid decreased with increasein. Pps activity, succinic acid either remained constant (0 μM IPTG) orincreased (10.50 μM IPTG) with an increase in Pps activity. Thisincrease could be contributed to Pps induced increase in PEP level,which is spilled over through PEP carboxylase and eventually tosuccinate.

EXAMPLE 1

Production of DAHP

This example demonstrates that the E. Coli AB2847 is unable to utilizeDAHP, and accumulates DAHP in the medium if DAHP synthase isoverexpressed. This strain was used as a host for detecting the fluxcommitted to the aromatic pathways. Since Draths et al. (Draths, K. M.,D. L. Pompliano, D. L. Conley, J. W. Frost, A. Berry, G. L. Disbrow, R.J. Staversky, and J. C. Lievense, “Biocatalytic synthesis of aromaticsfrom D-glucose: The role of transketolase,” J. Am. Chem. Soc., 1992,114, 3956-3962) have shown a possible limitation in the production ofDAHP by E4P, pATI (containing both aroG^(fbr) and tktA) was transformedinto AB2847 to eliminate the limitation of E4P. To test whether PEPsupply limits DAHP production, PEP synthase (Pps) was overexpressed inAB2847/pAT1 by transforming plasmid pPS341 into this strain. 20-70copies of the pps gene were expressed in the host cells. As a control,pPS341 was substituted by pPS341×1, which encodes an inactive, butstable pps gene product. The use of the inactive Pps control alloweddiscrimination between the effect of Pps activity and that of proteinoverexpression.

AB2847/pAT1/pUHE23-2 and AB2847/pAT1 were also used without any otherplasmid as additional controls to identify the effect of the cloningvector, pUHE23-2, on DAHP production.

As described above, the strains were grown in a rich medium (YE) withIPTG and re-suspended in a minimal medium. Since the overexpression ofPps under glycolytic conditions may cause growth inhibition,re-suspension cultures were used to minimize the effect of cell growthon the biocatalytic conversion. After re-suspension, the excreted DAHPand residual glucose were measured periodically. At 27 hours afterre-suspension, samples were taken for Pps and AroG assays. FIG. 1A showsthat the strain overexpressing active Pps increased the DAHP productionby almost two-fold. The strains containing pPS341×1 or pUHE23-2 producedthe same amount of DAHP as the one containing only pAT1. FIG. 1B showsthat, as expected, Pps activity was ten-fold overexpressed in the straincontaining pPS341, while the aroG activity in all strains remain almostconstant. These data strongly suggest that the activity of Pps isresponsible for the increase in the DAHP production, whereas theinactive Pps or the cloning vector has no observable effect on DAHPproduction.

The specific glucose consumption rates of these strains were notinfluenced by the presence of active or inactive Pps, nor by the cloningvector (data not shown). Therefore, the strain overexpressing Pps showedalmost a two-fold increase in overall DAHP yield (c.a. 90% molar) ascompared to the controls (ca. 52% molar), suggesting that Pps improvesboth the productivity and the yield of DAHP production. The maximumtheoretical yield from glucose to DAHP is 86%, which is slightly lowerthan the measured yield from the strain overexpressing Pps. Because bothglucose and DAHP measurements were reasonably reproducible, thediscrepancy may be attributed to the inaccuracy of the extinctioncoefficient used to calculate DAHP concentration. However, theextinction coefficient has been calibrated by biosynthesized DAHP fromcell extract and known amounts of E4P and PEP. Results show that theextinction coefficient is roughly within 30% accuracy. Therefore, theyield of DAHP is reasonably close. to the theoretical maximum, eventhough it may and probably; is lower than the theoretical value.

To determine whether the Pps effect requires overexpressed transketolase(Tkt) as well, plasmid pRW5, which contains only aroG^(fbr), was used inplace of pAT1 in the above experiments. It was found that overproductionof Pps did not increase the DAHP production (FIG. 2A) without theelevated Tkt activity. Therefore, as limitation of small molecules inthe biosynthesis of DAHP is concerned, the first limitation arises fromthe supply of E4P. This bottleneck shifts to the supply of PEP when Tktis overexpressed, which is believed to increase the supply of E4P.

EXAMPLE 2

As shown above, Pps overexpression improved DAHP production fromglucose. We were interested to know whether the basal level of Ppsexpression in glucose medium contributed to the production of DAHP.Therefore, the chromosomal pps gene in strain AB2847 was knocked out.The resulting strain (JCL1362) was used as the host to repeat the aboveexperiments. Results show that inactivation of chromosomal pps did notsignificantly affect the DAHP production in strains containing pRW5 orpAT1 (FIG. 2B). Therefore, the basal level of pps expression in glucosemedium did not contribute to the DAHP production.

Since PEP is also converted to OAA by Ppc, the deletion of this enzymemay increase the supply of PEP. Therefore, the ppc gene on thechromosome of AB2847 was inactivated to determine whether DAHPproduction could be increased without Pps overexpression. This was doneby transducing AB2847 with a PI lysate grown on JCL1242 ppc::Km. Theresulting transductant, JCL1283 aroB ppc::Km was then transformed withpAT1 or pRW5 and tested for DAHP production in the re-suspension cultureas described above. To avoid limitation of OAA in the ppc strain, theculture medium was supplemented with succinate, which was shown to haveno effect on DAHP production, (data not shown). Contrary to theexpectation, ppc mutation did not increase the production of DAHP (FIG.2B), suggesting that the metabolic flux from PEP to OAA was notsignificant under the experimental conditions tested here. In fact, theppc mutation actually decreased the DAHP production for unknown reasons.

EXAMPLE 3

Production of Tryptophan

Existing technologies for the production of tryptophan utilize eithernaturally occurring microorganisms, mutated microorganisms, orgenetically engineered microorganisms. These microorganisms include, butare not limited to Escherichia coli, Brevibacteria, Corynebacieria, andyeast. The altered pathways may include: (1) deletion of pathwaysbranching off to phenylalanine and tyrosine; (2) deletion of pyruvatekinases (pyk); (3) deletion of PEP carboxylase (ppc), (4) deletion ofphosphoglucose isomerase (pgi); (5) desensitize feed-back inhibition ofenzymes in the chorismate pathway and the trp operon; (6) deletion ofthe repressor, trpR, and the attenuation sequence in the trp operon, (7)deletion of tryptophan degradation enzymes; (8) overexpression of thetrp operon enzymes; (9) overexpression of the wild-type or feedbackresistant AroF, AroG, or AroH, or any enzyme in the chorismate pathway;(10) overexpression of SerA; and (11) overexpression of TktA or TktB.

To produce tryptophan, strain ATCC31743 which contains chromosomalmarkers such as trpR Δ(trpAE) tna can be used as a host. This strainalso contains a plasmid pSC102trp which harbors trpAE operon. PlasmidspAT1 and pPS341 (or pPS706 or pPSL706) can be transformed into thisstrain. The serA gene can be cloned into any of the plasmids.Alternatively, these cloned genes (trpAE, aroG, tktt, pps or serA) canbe consolidated to one or two plasmids. The resulting strain was grownin MT medium which contains, per liter: KH₂PO₄, 3 g: K₂HPO₄, 3g; K₂HPO₄,7 g; NH₄CL, 3 g; MgSO₄, 0.2 g; FeSO₄ 7H₂O), 10 mg, glucose, 0 to 30 g.

The Pps technology is compatible with all of the above alterations inmetabolism. Alterations that favor the supply of E4P, such as thedeletion of phosphoglucose isomerase, may eliminate the need foroverexpression Tkt associated with Pps in the preferred embodiment.Higher AroG levels may also eliminate the need for overexpressing Tkt.The Pps technology can be used in microorganisms such as Brevibacteriaand Corynebacteria.

EXAMPLE 4

Production of Phenylalanine

Pathway alterations for the production of phenylalanine are simnilar tothe above except at the terminal pathways leading to phenylalanine.These include (1) the overexpression of the enzymes from chorismate tophenylalanine; (2) deletion of trip operon; and (3) deletion ofphenylalanine degrading enzymes, and (4) desensitize all the enzymesfrom DAHP to phenylalanine so that they are not inhibited by the latter.

To produce phenylalanine, an E. coli mutant (W3110 Δtrp Δtyr Δphe) (ref:Forberg, Eliaeson, and Haggstrom, 1988) can be used as a host. PlasmidspAT1 and pPS341 (or pPS706, pPSL706) can then be transformed into thisstrain. In addition, the pheAfbr gene from plasmid pJN6 (same ref) canbe cut and ligated into either pPS341 or pAT1. The resulting stain canbe cultured in the following medium containing, per liter; NH₄CL, 5 g;K₂SO₄, 0.8 g; KH₂PO₄, 0.5 g; Na₂HPO₄, 1 g; Na-citrate, 2.5 g; FeCL₃6H₂O, 0.01 g; CaCl₂ 2H₂O, 0.20; MgCl₂ 6H₂O, 0.8 g; tryosine, 0.05 g;tryptophan, 0.025 g, glucose 10-30 g.

EXAMPLE 5

Production of Tyrosine

Pathway alterations for the production of tyrosine are similar to theabove except at the terminal pathways leading to tyrosine. These include(1) the overexpression of the enzymes from chorismate to tyrosine; (2)deletion of trp operon and the phenylalanine branch; (3) deletion oftyrosine degrading enzymes; and (4) desensitize all the enzymes fromDAHP to tyrosine so that they are not inhibited by the later.

EXAMPLE 6

Production of indigo

Production of indigo may be achieved by converting tryptophan or anintermediate from the trp pathway to indigo, either in vitro or in vivo.Since the Pps technology increases the production of DAHP, it will alsoincrease the supply of any metabolite that serve as the precursor forindigo. To produce indigo, the tryptophan producing strain describedabove can be used as a host. However, the strain needs to be made tna+and overexpressing naphthalene dioxygenase from Pseudomonas putida. Inthis strain, tryptophan produced will be degraded by tryptophanase toindole, which is then converted to cis-indole-2, 3-dihydrodiol by thecloned naphthalene dioxygenase. The cis-indole-2, 3-dihydrodiol producedis spontaneously converted to indigo in the presence of oxygen. ATCC31743 is the strain used in conversion of DAHP to tryptophan.

EXAMPLE 7

Production of Quinoid Organics

Quinoid organics can be derived fromr dehydroquinate which is adown-stream metabolite of DAHP. To produce quinic acid, E. coli AB2848aroD harboring pTW8090A which contains the gene qad (quinic aciddehydrogenase from Klebsiella pneuinoniae) (ref: Draths, Ward, andFrost, 1992, JACS, 114, 9725-9726), and pKD136 (ref: same as above)which contains tkt, aroF, and aroB genes can be used as a host. The ppsgene can be cloned into one of these plasmids and be simultaneouslyoverexpressed. It has been reported that at least 80 mM of D-glucose canbe converted into 25 mM of quinic acid. After cell removal, quinic acidin the supernatant can be converted into benzoquinone after addition ofsulfuric acid and technical grade manganese (IV) dioxide and heating at100° C. for 1 h. In the absence of acidification, aqueous solutions ofpurified quinic acid were converted. to hydroquinone in 10% yield uponheating at 100° C. for 18 h with technical grade manganese dioxide.

EXAMPLE 8

Production of Catechol

Production of catechol may be achieved by transforming pps into E. Coliexpressing pKD136. Since the Pps technology increases the production ofDAHP, it will also increase the supply of any metabolite that serves asthe precursor for catechol.

EXAMPLE 9

Characterization of luxI′-driven pps expression

To characterize the luxI′-driven pps expression, pPS706 were transformedinto AB2847/pRW5 and AB2847/pAT1, and the Pps activity was measured whenthe strains were cultured in either glucose or xylose medium. The Ppsactivity increased with the autoinducer (N-(3-xox-hexanoyl) -hmoserinelactone) concentration and reached saturation when 1 μM autoinducer wasused. Pps activity in the same strain growing in xylose medium was thesame as that in glucose. The Pps activity was independent of IPTGconcentration (data not shown). Therefore, the inventor achieved theindependent modulation of three key enzymes in the production ofaromatics: AroG (IPTG-inducible), TktA (on/off or presence/absence) andPps (autoinducer-inducible).

While in accordance with the patent statutes, the best mode andpreferred embodiments of the invention have been described, it is to beunderstood that the invention is not limited thereto, but rather is tobe measured by the scope and spirit of the appended claims.

What is claimed is:
 1. A method for increasing 3′deoxy-D-arabino-heptulosonate (DAHB) production relative to wild typehost cells, comprising the steps of: (a) transforming a genetic agentinto a microorganism to overexpress Pps to convert pyruvate tophosphoenolpyruvate (PEP); and (b) cultivating the transformed organismin a medium.
 2. The method of claim 1 further comprising the step ofisolating the DAHP.
 3. The method of claim 1 further comprising the stepof overexpressing the tkt gene.
 4. The method of claim 1 wherein themicroorganism is Escherichia coli AB2847.
 5. The method of claim 1further comprising the step of transforming a genetic agent into amicroorganism to overexpress Tkt.
 6. A method for producing cyclicmetabolites comprising the steps of: (a) transforming a genetic agentinto a microorganism to overexpress Pps wherein the microorganism is agenetically engineered strain adapted to produce a desired product; (b)cultivating the transformed microorganism in a nutrient containingmedium.
 7. A process for the production of DAHP which comprisescultivating a microorganism in a nutrient medium and overexpressing thePps gene.
 8. The process of claim 7 wherein the step of overexpressingthe Pps gene comprises transforming a plasmid selected from the groupconsisting of pPS341, pPSL706, and pPS706 into the microorganism.
 9. Theprocess of claim 7 further comprising the step of overexpressing the tktgene.
 10. The process of claim 9 wherein the step of overexpressing theTkt gene comprises transforming the plasmid pAT1 into the microorganism.